Contractile function of cardiac muscle is strongly dependent on fine regulation of intracellular ion levels. The primary aim of this project is to increase the understanding of the transsarcolemmal transport of sodium and calcium by the Na-Ca exchange mechanism. The physiological significance of Na-Ca exchange is controversal, and the long range objective is to relate molecular findings to intact preparations. Five approaches are proposed as follows: 1. Identification and isolation of the Na-Ca exchange protein. Molecular characterization of the Na-Ca exchanger requires that the protein first be isolated. Excellent progress towards this goal has been achieved, and several approaches to complete the purification procedure are planned. 2. Expression of Na-Ca exchange in Xenopus oocytes. To begin to examine the molecular biology of Na-Ca exchange, cardiac mRNA is being injected into oocytes to induce expression of exchange activity. Experiments are then planned to fractionate mRNA and, eventually, to clone the Na-Ca exchange protein. 3. Interaction of the Na-Ca exchanger with the membrane lipid environment. Na-Ca exchange activity is very sensitive to changes in the lipid environment of native sarcolemmal vesicles. Lipid-exchanger interactions are now being explored in more detail using solubilization/reconstitution techniques. 4. Kinetics of Na-Ca exchange. Much basic characterization of Na-Ca exchange is planned using both native and reconstituted cardiac sarcolemmal vesicles. For example, stoichiometry, effects of intravesicular Ca, and Na-Ca exchange will be examined. 5. Long term regulation of Na-Ca exchange. The response of cultured neonatal heart cells to chronic changes in intracellular ion levels will be examined. For examples, Na-Ca exchange will be measured in sarcolemmal vesicles isolated from control cells and cells which have been exposed to ouabain for 24 hours. It is speculated that the cells will adjust rates of synthesis and degradation of different ion transporters in response to interventions.